This NIAID K22 Career Transition Award focuses on the long-term research goals of the candidate, Nitya Jain, Ph.D., to become an independent investigator and to generate additional data for a future R01 application. Dr. Jain is committed to this career path and has extensive experience in cellular and molecular immunology. The proposal outlined here addresses the impact of postnatal intestinal microbial colonization on thymic T cell development. This is a significant area of research related to pediatric immunology. The immune system of the newborn faces unique challenges in the period immediately following birth. In lieu of minimal antigenic exposure in utero to stimulate adaptive immunity, the newborn's immune system relies heavily on the innate immune effectors for protection against infections. However, cells of the adaptive immune system are rapidly maturing and proliferating in the thymus at the same time as the microbiota are maturing and dividing in the intestine of the newborn. Thus, the developing T cells of the infant immune system face the monumental task of recognizing and classifying the burgeoning gut microbiota as benign, commensal or pathogen. Loss of tolerance to and alterations in intestinal microbiota has been associated with onset of autoimmune T1D and Inflammatory Bowel Disease in adults. Thus, an important question remains, whether and how the developing T cells in the neonate are influenced by microbial colonization events in the intestine. The studies described in this proposal will address this knowledge deficit. The long-term objective of this proposal is to understand the influence of intestinal microbial colonization on adaptive T cell development and function. The specific objective of this proposal is to identify molecular mediators of thymic-intestine crosstalk. We propose that the changing microbial environment in the intestines influences developing T cells in the thymus and impacts immune responses in adulthood. We will test this hypothesis in two Specific Aims. In Aim 1, we will comprehensively characterize the impact of intestinal microbial colonization on neonatal thymic T cell development using germ-free and monocolonized germ-free mice. In Aim 2, we will determine the microbiota-dependent molecular signal that mediates thymic-intestinal crosstalk. The data obtained from these aims will lead to future studies that will further elaborate on the unique nature of postnatal immunity and identify specific pathways that could be manipulated to direct postnatal immune responses towards desired outcomes.